This is a project to continue studies which emphasize understanding of initial events in Mycobacterium avium complex (MAC) infection. Our work to this point has led to two fundamental discoveries. First, we have shown that MAC organisms bind fibronectin (Fn) via fibronectin attachment protein (FAP) and that this binding is essential for efficient invasion of epithelial cells, which is likely to be a critical component of initial infection by MAC. Our second fundamental discovery was that, unlike epithelial cell invasion, FN opsonization of MAC did not lead to infection of macrophages. Instead, macrophage invasion depended on complement activation. In particular we showed that a fragment of complement activation called C2a, normally inactive, could work together with a MAC co-factor to cleave macrophage-derived C3 and thus provide a recognition signal for macrophage uptake of MAC. It is very important that M. tuberculosis, M. leprae, and M. bovis BCG share the ability to use C2a to invade macrophages with MAC, but the fast growing mycobacteria such as M. vaccae, M. smegmatis and M. phlei, which are not intracellular pathogens, do not. This suggests that this property is a virulence factor conserved among intracellular pathogenic mycobacteria. Based on these results we propose to i) determine the significance of MAC interaction with epithelial cells in infection; and ii) establish role of complement in MAC infection in vivo. To do this, we will use a variety of biochemical and cell and molecular biologic approaches to understand FAP-Fn binding during establishment of systemic infection; FAP biosynthesis, secretion, and interaction with MAC cell wall, the mechanism of inhibition of FAP- mediated invasion of epithelial cells by integrin alpha4beta1, and to characterize the MAC glycolipid which activates complement C2a. Finally we will use genetically mutant mice to determine the importance of complement for in vivo infection. These studies will build on our previous work to provide a very complete picture of the initial events in MAC infection. These data will facilitate through a thorough evaluation of potentially important targets for therapeutic intervention.